Bacterium

ABSTRACT

The present invention relates to a bacterial cell with texturizing property, starter cultures comprising the cell, and dairy products fermented with the starter culture.

This application is a 371 of PCT/EP2011/051239, filed Jan. 28, 2011,which claims foreign priority to the following five Danish applications:PA2010 00070, filed Jan. 28, 2010, PA 2010 00081, filed Jan. 29, 2010,PA 2010 00519, filed Jun. 11, 2010, PA 2010 00894 filed Oct. 1, 2010,and PA 2010 01051 filed Nov. 19, 2010. This application also claimspriority to PCT/EP2010/062808 filed Sep. 1, 2010.

FIELD OF INVENTION

The present invention relates to a bacterial cell which has improvedphage resistance, starter cultures comprising the cell, and dairyproducts fermented with the starter culture.

BACKGROUND OF INVENTION

The food industry uses numerous bacteria, in particular lactic bacteria,in order to improve the taste and the texture of foods but also in orderto extend the shelf life of these foods. In the case of the dairyindustry, lactic bacteria are used intensively in order to bring aboutthe acidification of milk (by fermentation) but also in order totexturize the product into which they are incorporated.

Among the lactic bacteria used in the food industry, there can bementioned the genera Streptococcus, Lactococcus, Lactobacillus,Leuconostoc, Pediococcus and Bifidobacterium. The lactic bacteria of thespecies Streptococcus thermophilus are used extensively alone or incombination with other bacteria for the production of food products, inparticular fermented products. They are used in particular in theformulation of the ferments used for the production of fermented milks,for example yogurts. Certain of them play a dominant role in thedevelopment of the texture of the fermented product. This characteristicis closely linked to the production of polysaccharides. Among thestrains of Streptococcus thermophilus it is possible to distinguishtexturizing and non-texturizing strains.

WO2007095958A1 discloses Streptococcus thermophilus strains withtexturizing properties. In FIG. 1 it can be seen that the mosttexturizing strain CHCC8833 (DSM17876) has a shear stress value ofaround 59 Pa.

In order to meet the requirements of the industry, it has becomenecessary to provide novel texturizing strains of lactic bacteria, inparticular of Streptococcus thermophilus, for texturizing food products.Especially there is a need for a novel texturizing strain ofStreptococcus thermophilus which can be used together with a strain of aLactobacillus species. Another need of the industry is that the strainis resistant to bacteriophages normally found in the food industry.

SUMMARY OF INVENTION

The present inventors have provided a novel group of lactic acidbacteria of the species Streptococcus thermophilus and Lactobacillusbulgaricus, which surprisingly is more resistant to phage attack thanthe (mother) strain from which it is obtained. Further, it hassurprisingly turned out that this group of bacteria generates highershear stress and/or gel stiffness than the mother strain when thebacteria are used for fermenting milk.

It is surprising that a phage resistant mutant strain generates moretexture, e.g. higher shear stress and/or gel stiffness, (when the strainis used to ferment milk) than the mother strain, and it is especiallysurprising that a phage resistant mutant strain of a strain which (also)contains a mutation in the galK gene (relative to the wildtype strain)generates more texture (when the strain is used to ferment milk) thanthe mother strain.

In accordance with the above surprising findings, the present inventionrelates to a method for manufacturing a texturizing lactic acidbacterium strain by screening for phage resistant mutants of a motherstrain, e.g. a method for manufacturing a texturizing lactic acidbacterium (e.g. a bacterium which, besides being e.g. phage resistant,substantial phage resistant, and/or possesses increased phage resistancecompared to the mother stain, is more texturizing than the motherstrain), comprising the steps:

a) Providing a lactic acid bacterial strain (the mother strain); and

b) Isolating a mutant strain of the mother strain, which mutant strainis resistant against more phages (e.g. more types of phages or morestrains of phages) than the mother strain.

Further, the present invention relates to texturizing lactic acidbacteria strains, such as S. thermophilus strains or Lactobacillusbulgaricus stains, which are modified to be resistant to more phagesthan the mother strain, in particular against one of the phages CHPC658,CHPC1057, CHPC1089 and CHPC1152.

DETAILED DISCLOSURE

In a first aspect, the present invention relates to a method formanufacturing a lactic acid bacterium (which is e.g. phage resistant,substantial phage resistant, and/or possesses increased phage resistancecompared to the mother stain, or which generates higher shear stressand/or gel stiffness than the mother strain when the bacteria are usedfor fermenting milk), comprising the steps:

-   -   a) Providing a lactic acid bacterial strain (the mother strain);        and    -   b) Isolating a mutant strain of the mother strain, which mutant        strain is resistant against more phages (e.g. more types of        phages or more strains of phages) than the mother strain, and/or        which mutant is resistant against a phage which the mother        strain is not resistant to (under that same conditions).

In an interesting aspect, the present invention relates to a method formanufacturing a lactic acid bacterium (which is e.g. phage resistant,substantial phage resistant, and/or possesses increased phage resistancecompared to the mother stain and/or which generates higher shear stressand/or gel stiffness than the mother strain when the bacteria are usedfor fermenting milk), comprising the steps:

-   -   a) Providing a lactic acid bacterial strain (the mother strain);        and    -   a1) Exposing the lactic bacterial strain to a bacteriophage,        e.g. a bacteriophage which is able to lyse the mother strain,        such as a phage selected from the group consisting of CHPC658,        CHPC1057, CHPC1089 and CHPC1152, and;    -   b) Isolating a mutant strain of the mother strain, which mutant        strain is resistant against the phage (or which strain is not        lysed by the phage).

Also, the present invention relates to a method, comprising the steps:

-   -   a) Providing a lactic acid bacterial strain (the mother strain);    -   a1) Exposing the lactic bacterial strain to a bacteriophage,        e.g. a bacteriophage which is able to lyse the mother strain,        such as a phage selected from the group consisting of CHPC658,        CHPC1057, CHPC1089 and CHPC1152;    -   a2) Incubating the exposed bacterial cells in a growth medium;        and    -   b) isolating a mutant strain of the mother strain, which mutant        strain is not lysed by the bacteriophage

The methods of the invention may comprise the step

-   -   c) mutating (e.g. by chemical treatment or radiation treatment,        or by means of genetic engineering techniques) the mother        strain, e.g. before, during or after step a1).

Also, any method of the invention may comprise the step

-   -   d) introducing a mutation in the galK gene or the galK        regulatory sequence (e.g. promoter) of the strain (e.g. by        chemical treatment or radiation treatment, or by means of        genetic engineering techniques), e.g. before, during or after        step c), or before, during or after step a1).

In an interesting embodiment, a method of the invention comprises thesteps:

-   -   Providing a lactic acid bacterial strain (the mother strain);    -   mutating (e.g. by chemical treatment or radiation treatment, or        by means of genetic engineering techniques) the mother strain;    -   Exposing the resulting lactic bacterial strain to a        bacteriophage, e.g. a bacteriophage which is able to lyse the        mother strain, such as a phage selected from the group        consisting of CHPC658, CHPC1057, CHPC1089 and CHPC1152;    -   Incubating the exposed bacterial cells in a growth medium; and    -   isolating a mutant strain of the mother strain, which mutant        strain is not lysed by the bacteriophage;

The method of the invention may lead to that a mutation is introduced inthe promoter region of the galK gene, such as in the −10 region (thePribnow box) or in the region between the Pribnow box and the ribosomebinding site.

Also, the method of the invention may lead to a mutation resulting inincreased galactose fermenting activity compared to the mother strain.

The mutation may result in the replacement of one or more nucleotides inthe region between the Pribnow box and the ribosome binding site of thegalK gene, such as replacement of C in 10 the sequence TTCAGT downstreamfrom the wildtype Pribnow box with a nucleotide selected from the groupconsisting of A, T and G.

In an other embodiment, the mutation may result in:

-   -   the replacement of one or more nucleotides in the region between        the Pribnow box and the ribosome binding site of the galK gene,        such as replacement of C in the sequence TTCAGT (SEQ ID NO:6)        downstream from the wildtype Pribnow box with a nucleotide        selected from the group consisting of A, T and G; and/or    -   the replacement of one or both of C and G in the wildtype −10        region (TACGAT, SEQ ID NO:7) with a nucleotide independently        selected from the group consisting of A and T; and/or    -   the replacement of C the wildtype −10 region (TACGAT, SEQ ID        NO:7) with a nucleotide independently selected from the group        consisting of A and T; and/or    -   the replacement of C the wildtype −10 region (TACGAT, SEQ ID        NO:7) with T; and/or    -   a −10 region which has the nucleotide sequence TATGAT (SEQ ID        NO:8), TATTAT (SEQ ID NO:9) or TACTAT (SEQ ID NO:10).

It should be understood that the mother strain used in a method of theinvention may be a gal+ strain, preferable a strain which is able toreduce pH by a value of at least 1.0 after 16 hours incubation at 37degrees C. in M17 with 2% galactose (galactose added as solecarbohydrate), inoculated in an amount of at least 10E4 cells pr ml ofmedium. Examples on such a strain is a strain wherein:

-   -   one or more nucleotides in the region between the Pribnow box        and the ribosome binding site of the galK gene has been        replaced, such as replacement of C in the sequence TTCAGT (SEQ        ID NO:6) downstream from the wildtype Pribnow box with a        nucleotide selected from the group consisting of A, T and G;        and/or    -   one or both of C and G in the wildtype -10 region (TACGAT, SEQ        ID NO:7) has been replaced with a nucleotide independently        selected from the group consisting of A and T; and/or    -   C in the wildtype −10 region (TACGAT, SEQ ID NO:7) has been        replaced with a nucelotide independently selected from the group        consisting of A and T; and/or    -   C in the wildtype −10 region (TACGAT, SEQ ID NO:7) has been        replaced with T; and/or    -   the −10 region has the nucleotide sequence TATGAT (SEQ ID NO:8),        TATTAT (SEQ ID NO:9) or TACTAT (SEQ ID NO: 10). Novel gal+        strains is a part of the present invention.

The method of the invention may comprise one or more further step(s)selected from the group consisting of:

-   -   c1) screening for a mutant strain having phage resistance, such        as increased phage resistance compared to the mother stain; and    -   c2) screening for a mutant strain having Gal+ phenotype, such as        increased galactose degrading activity compared to the mother        stain.

An embodiment of the method of the present invention relates to a methodfor manufacturing a lactic acid bacterium, comprising the steps:

-   -   Providing a lactic acid bacterial strain (the mother strain);    -   optionally mutating the mother strain;    -   Exposing the optionally mutated mother strain to a        bacteriophage, e.g. a bacteriophage which is able to lyse the        mother strain, such as a phage selected from the group        consisting of CHPC658, CHPC1057, CHPC1089 and CHPC1152;    -   optionally incubating the exposed bacterial cells with a growth        medium; and    -   screening for a mutant strain having phage resistance, such as        increased phage resistance compared to the mother strain; and/or        screening for a mutant strain having Gal+ phenotype, such as        increased galactose degrading activity compared to the mother        stain.

The mutant strain obtained by a method of the invention may be aspontaneous mutant, a mutant obtained by mutagenesis of the motherstrain by means of e.g. chemical treatment or radiation treatment, or amutant obtained by means of genetic engineering techniques. Aninteresting mutant of the invention is a mutant of CHCC6008, esp. amutant which is gal+ and resistant against CHCP1152.

In an embodiment of the method of the invention, the mutant strain isphage resistant, substantial phage resistant, and/or possesses increasedphage resistance compared to the mother stain, and wherein the phage isselected from the group consisting of: a bacteriophage which is able tolyse the mother strain, CHPC658, CHPC1057, CHPC1089 and CHPC1152.

It is presently preferred that the bacterium (the mother strain) isselected from a species selected from the group consisting ofLactococcus spp., Streptococcus spp. (e.g. Streptococcus thermophilus),Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp.,Propionibacterium spp, and Bifidobacterium spp.

In an interesting embodiment of a method of the invention, the bacterium(the mother strain) is selected from a strain which has one or morefeatures selected from the group consisting of: ability to texturizemilk, ability to produce a polysaccharide, such as an exopolysaccharideor a capsular polysaccharide, ability to create viscosity when incubatedin milk, and ability to increase shear stress when incubated in milk.

In another aspect, the present invention relates to a lactic acidbacterium or strain which is obtainable by a method of the invention.

In a further aspect, the present invention relates to a lactic acidbacterium or strain, such as a bacterium or strain obtainable by amethod of the invention, which generates a viscosity in fermented milkgreater than about 70 Pa (pascal) (such as greater than 73 Pa, greaterthan 77 Pa or greater than 79 or greater than 80 Pa), measured as shearstress after 12 hours of 15 growth at 37 degrees C., e.g. inoculated inan amount of at least 10E4 cells pr ml of milk. It is believed that itis possible to obtain strains which generates a viscosity in fermentedmilk up to 100 Pa, up to 120 Pa, up to 150 Pa, or even up to 200 Pa.Examples of ranges for viscosity obtainable are: 70 to 200 Pa, 75 to 150Pa, 78 to 120 Pa, 79 to 100 Pa, and 80 to 90 Pa. Especially, the presentinvention relates to a strain of S. thermophilus, which is able togenerate a viscosity of 79 to 100 Pa in fermented milk.

Examples of a strain of the invention is a bacterial strain belonging tothe species Streptococcus thermophilus, selected from the groupconsisting of: CHCC11342 (DSM 22932), CHCC11977 (DSM22935), CHCC12339(DSM24090), and CHCC13140 (DSM 24023), and mutants and variants of anyof these. In addition, the invention relates to all novel strainsmentioned herein, as well as their mutants and variants. Specifically,the present invention relates to the strain CHCC11977, and a mutantthereof.

In a further aspect the present invention relates to a lactic acidbacterium or strain of the invention, which generates a shear stress infermented milk greater than about 70 Pa (such as greater than 73 Pa,greater than 77 Pa or greater than 80 Pa), measured after 12 hours ofgrowth at 37 degrees C., e.g. inoculated in an amount of at least 10E4cells pr ml of milk. The bacterium or strain may belong to the speciesStreptococcus thermophilus or Lactobacillus delbrueckii subsp.bulgaricus.

The present invention also relates to a bacterial strain belonging tothe species Lb delbrueckii sbsp. bulgaricus, such as a strain selectedfrom the group consisting of CHCC12813 (DSM24074) and CHCC12841, andmutants and variants thereof. Interesting strains are those whichgenerates a shear stress in fermented milk greater than about 60 Pa(such as greater than 65 Pa, greater than 69 Pa or greater than 72 Pa),measured after 12 hours of growth at 37 degrees C., e.g. inoculated inan amount of at least 10E4 cells pr ml of milk. Especially interestingare those generating a shear stress in fermented milk in the range from60 to 100 Pa (such as from 65 to 90 Pa, from 69 to 85 Pa or from 72 to80 Pa), measured after 12 hours of growth at 37 degrees C., e.g.inoculated in an amount of at least 10E4 cells pr ml of milk.

In a still further aspect the present invention relates to a lactic acidbacterium or strain or mutant or variant of the invention, whichgenerates a gel stiffness in fermented milk greater than about 110 Pa(such as greater than 115, greater than 120 or greater than 125 Pa),measured after 12 hours of growth at 37 degrees C., e.g. inoculated inan amount of at least 10E4 cells pr ml of milk. It is presentlypreferred that the gel stiffness is within the range 110 to 200 Pa, ormore preferred within the range 120 to 190 Pa or 125 to 180 Pa. Thebacterium or strain may belong to the species Streptococcus thermophilusor Lactobacillus delbrueckii subsp. bulgaricus. Especially, the presentinvention relates to a strain of S. thermophilus, which is able togenerate a gel stiffness of 125 to 175 Pa in fermented milk.

In an interesting embodiment, the present invention relates to a lacticacid bacterium or strain, which belongs to the species Lb. delbrueckiisbsp. bulgaricus. Interesting examples on this embodiment are abacterium or strain, which generates a viscosity in fermented milkgreater than about 60 Pa (such as greater than 65 Pa, greater than 69 Paor greater than 72 Pa), measured as shear stress after 12 hours ofgrowth at 37 degrees C., e.g. inoculated in an amount of at least 10E4cells pr ml of milk, and/or a bacterium or strain, which generates aviscosity in fermented milk in the range from 60 to 100 Pa (such as from65 to 90 Pa, from 69 to 85 Pa or from 72 to 80 Pa), measured as shearstress after 12 hours of growth at 37 degrees C., e.g. inoculated in anamount of at least 10E4 cells pr ml of milk.

In an other aspect, the present invention relates to a compositioncomprising lactic acid bacteria or a strain of the invention, such asbacteria belonging to the strain CHCC11977 or a mutant thereof. It ispreferred that such composition comprises at least 10exp10 CFU (cellforming units) of said bacteria.

In an embodiment, the composition may comprise, either as a mixture oras a kit-of-parts,

-   -   a strain belonging to a Lactobacillus species, such as a        Lactobacillus delbrueckii subsp. bulgaricus (synonym:        Lactobacillus bulgaricus), a L. johnsonii, or a L. fermentum        strain; and    -   a strain of a lactic acid bacterium of the invention, such as a        strain belonging to the species Streptococcus thermophilus, e.g.        a strain selected from the group consisting of CHCC11342 (DSM        22932), CHCC11977 (DSM22935), CHCC12339, and CHCC13140 (DSM        24023), and mutants and variants of any of these,    -   such as a composition wherein the strain belonging to a        Lactobacillus species is a strain belonging to a polysaccharide        (such as a heteropolysaccharide, homopolysaccharide) and/or        fructosyl transferase enzyme producing Lactobacillus species.

The composition of the invention may comprise at least 10exp10 CFU (cellforming units) of a strain belonging to a Lactobacillus species; and/orat least 10exp10 CFU of a strain belonging to the species Streptococcusthermophilus.

In an interesting embodiment, the composition of the invention comprisesat least 10exp10 CFU (cell forming units) of a strain belonging to apolysaccharide (such as homopolysaccharide) and/or fructosyl transferaseenzyme producing Lactobacillus species; and at least 10exp10 CFU of astrain belonging to the species Streptococcus thermophilus.

The composition may be usable as a starter culture, and may be infrozen, freeze-dried or liquid form.

In a further aspect, the present invention relates to a method forproducing a fermented milk product, comprising fermenting a milksubstrate (such as cow's milk) with a lactic acid bacterium of theinvention, a strain of the invention, or a composition of the invention.

This method may further comprise fermenting the milk substrate with astrain belonging to a Lactobacillus species, such as a strain of L.bulgaricus or L. fermentum, e.g. a strain selected from the groupconsisting of CHCC10019, CHCC10935, or CHCC3984, and mutants andvariants of any of these strains. E.g., the milk substrate is fermentedwith a composition, strain or bacterium of the invention, such as astrain belonging to the species Streptococcus thermophilus before,during, or after the fermentation with a strain belonging to aLactobacillus species, or, the milk substrate is fermented with a strainor bacterium belonging to the species Streptococcus thermophilus duringthe fermentation with a strain belonging to a polysaccharide producingLactobacillus species.

The method of the invention for producing a fermented milk product maycomprise adding an enzyme to the milk substrate before, during and/orafter the fermenting, such as an enzyme selected from the groupconsisting of: an enzyme able to crosslink proteins, transglutaminase,an aspartic protease, chymosin, and rennet.

In yet another aspect, the present invention relates to a dairy product,such as a fermented milk product (e.g. yoghurt or buttermilk) or acheese (e.g. fresh cheese or pasta filata), obtainable by the abovemethod of invention. The fermented milk product may e.g. be astirred-type product, a drinkable product, or a set-type product. Thedairy product of the invention may optionally comprise an ingredientselected from the group consisting of: a fruit concentrate, a syrup, aprobiotic bacterial culture, a coloring agent, a thickening agent, aflavoring agent, and a preserving agent.

Thus, the present invention relates to a fermented milk productobtainable by the method of the invention, which optionally comprises aningredient selected from the group consisting of:

a fruit concentrate, a syrup, a probiotic bacterial culture, a coloringagent, a thickening agent, a flavoring agent, and a preserving agent;and/or which optionally is in the form of a stirred type product, a settype product, or a drinkable product.

Also the invention relates to a dairy product, which is made byfermenting a milk substrate (such as cow's milk) with a lactic acidbacterium of the invention (e.g. a strain belonging to the speciesStreptococcus thermophilus, such as DSM 22884) and a lactic acidbacterium a species selected from Lactobacillus bulgaricus andLactobacillus fermentum (such as CHCC10019 (DSM19252), CHCC3984(DSM19251) and CHCC2008 (DSM22584)).

In an interesting embodiment, a dairy product of the invention has aviscosity/texture of more than 100 Pa (such as more than 102 or morethan 104 Pa), measured as shear stress, e.g. after 12 hours growth at 37degrees C. in milk. In a presently preferred embodiment, the viscosityof more than 100 Pa is obtained by growth of the bacterial cells alone,but higher viscosity values can be obtained by addition of chemicalcompounds, such as starch, gelatine, a carrageenan, etc.

In an other embodiment, the dairy product of the invention has aviscosity/texture in the range of 100 to 200 Pa, such as in the range of100 to 150 Pa, or in the range of 105 to 125 Pa, measured as shearstress.

In a further embodiment, the dairy product of the invention is adrinkable product, e.g. drinking yoghurt.

The invention also relates to novel bacteriophages usable in a method ofthe invention, such as a bacteriophage selected from the groupconsisting of CHPC658 (DSM 23961), CHPC1057, CHPC1089 and CHPC1152 (DSM23994), and mutant and variants thereof, such as mutants and variantswhich are able to lyse strains mentioned herein, e.g. the strainCHCC6008.

Further, the invention relates to novel bacterial strains, useable asmother strains in a method of the invention, such as strains selectedfrom the group consisting of CHCC11342 (DSM22932), CHCC10019 (DSM19252),CHCC11379 (DSM 22884), CHCC11976 (DSM 22934), and mutants thereof.

As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of the strict anaerobic bacteria, bifidobacteria,i.e. Bifidobacterium spp., are generally included in the group of lacticacid bacteria. These are frequently used as food cultures alone or incombination with other lactic acid bacteria. Lactic acid bacteria,including bacteria of the species Lactobacillus sp. and Streptococcusthermophilus, are normally supplied to the dairy industry either asfrozen or freeze-dried cultures for bulk starter propagation or asso-called “Direct Vat Set” (DVS) cultures, intended for directinoculation into a fermentation vessel or vat for the production of adairy product, such as a fermented milk product. Such cultures are ingeneral referred to as “starter cultures” or “starters”.

The term “milk” is to be understood as the lacteal secretion obtained bymilking any mammal, such as cows, sheep, goats, buffaloes or camels. Ina preferred embodiment, the milk is cow's milk. The term milk alsoincludes protein/fat solutions made of plant materials, e.g. soy milk.

The term “milk substrate” may be any raw and/or processed milk materialthat can be subjected to fermentation according to the method of theinvention. Thus, useful milk substrates include, but are not limited to,solutions/suspensions of any milk or milk like products comprisingprotein, such as whole or low fat milk, skim milk, buttermilk,reconstituted milk powder, condensed milk, dried milk, whey, wheypermeate, lactose, mother liquid from crystallization of lactose, wheyprotein concentrate, or cream. Obviously, the milk substrate mayoriginate from any mammal, e.g. being substantially pure mammalian milk,or reconstituted milk powder.

Preferably, at least part of the protein in the milk substrate isproteins naturally occurring in milk, such as casein or whey protein.However, part of the protein may be proteins which are not naturallyoccurring in milk.

The term “milk” is to be understood as the lacteal secretion obtained bymilking any mammal, such as cows, sheep, goats, buffaloes or camels. Ina preferred embodiment, the milk is cow's milk.

Prior to fermentation, the milk substrate may be homogenized andpasteurized according to methods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. If homogenization is performed prior tofermentation, it may be performed so as to break up the milk fat intosmaller sizes so that it no longer separates from the milk. This may beaccomplished by forcing the milk at high pressure through smallorifices.

“Pasteurizing” as used herein means treatment of the milk substrate toreduce or eliminate the presence of live organisms, such asmicroorganisms. Preferably, pasteurization is attained by maintaining aspecified temperature for a specified period of time. The specifiedtemperature is usually attained by heating. The temperature and durationmay be selected in order to kill or inactivate certain bacteria, such asharmful bacteria. A rapid cooling step may follow.

“Fermentation” in the methods of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the methods of theinvention comprises conversion of lactose to lactic acid.

Fermentation processes to be used in production of fermented milkproducts are well known and the person of skill in the art will know howto select suitable process conditions, such as temperature, oxygen,amount and characteristics of microorganism(s) and process time.Obviously, fermentation conditions are selected so as to support theachievement of the present invention, i.e. to obtain a dairy product insolid or liquid form (fermented milk product).

The term “stirred type product” specifically refers to a fermented milkproduct which sustains a mechanical treatment after fermentation,resulting in a destructuration and liquefaction of the coagulum formedunder the fermentation stage. The mechanical treatment is typically butnot exclusively obtained by stirring, pumping, filtrating orhomogenizing the gel, or by mixing it with other ingredients. Stirredtype products typically but not exclusively have a milk solid non-fatcontent of 9 to 15%.

The term “set-type product” includes a product based on milk which hasbeen inoculated with a starter culture, e.g. a starter culture, andpackaged next to the inoculating step and then fermented in the package.

The term “drinkable product” includes beverages such as “drinkingyoghurt” and similar. The term “drinking yoghurt” typically covers amilk product produced by fermentation by the combination ofLactobacillus species and Streptococcus thermophilus. Drinking yoghurttypically has a milk solid non-fat content of 8% or more. Furthermore,the live culture count for drinking yoghurt drinks is typically at least10E6 cell forming units (CFU) pr ml.

“Drinkable product” according to the present invention include anydrinkable product based on acidified milk substrates, thus includingfermented milk drinks and liquid yoghurt drinks. In the methods of thepresent invention, acidification is performed as a fermentation with amicroorganism, optionally an acid is added, such as an organic acid(e.g. lactic acid, lactobionic acid or GDL).

Drinkable products according to the invention are drinkable in the sensethat they are in liquid form and consumed as beverages, i.e. they aresuitable for drinking instead of being eaten with a spoon. “In liquidform” means that the products are in the fluid state of matter thusexhibiting a characteristic readiness to flow. Thus, the shape of aliquid is usually determined by the container it fills, in contrary toe.g. a gel-like substance, which is soft, but not free flowing, such ase.g. yoghurt or pudding. Drinkable products according to the inventionmay have a viscosity allowing the consumer to drink the products using astraw if desired.

An drinkable product according to the present invention may have a pH ofless than 4.6, preferably less than 4.4, more preferably less than 4.2and even more preferably about pH 4 or less. In one aspect, thedrinkable product has a pH of less than 3.8, such as less than 16.

An drinkable product according to the invention may have a fat contentof 0 to 2%, preferably below 1.5%, below 1% or below 0.5%, morepreferably of about 0.1% or less. The drinkable product may have a milksolid non-fat content of less than 20%, preferably less than 8.5%, lessthan 8%, less than 7.5%, less than 7%, less than 6.5% or less than 6%,and more preferably of about 5%.

An drinkable product according to the invention may have a proteincontent of between 0.5 and 4%. In one preferred aspect, the drinkableproduct has a protein content of below 1%. In another preferred aspect,the drinkable product has a protein content of between 2% and 3%.

An drinkable product according to the invention may have a shelf life ofmore than 7 days, preferably more than 14 days, more preferably morethan 28 days, such as more than 3 months.

An drinkable product according to the present invention may have animproved sedimentation stability. The stability may be determined afterhaving stored the drinkable product for an appropriate number of days bymeasuring the height of the whey collecting on the surface because ofsyneresis. It may also be determined after accelerated syneresis, suchas by centrifugation.

For a drinkable product, e.g. drinking yoghurt, a high shear treatment(e.g. homogenization) after fermentation is normally needed to breakdown the protein network in order to obtain smooth, homogeneous anddrinkable products. The breakdown of the network implies that drinkingyoghurts have a reduced sedimentation stability, resulting insedimentation of protein to the bottom during shelf life. High fatrevels and high protein content increase sedimentation stability, whilelow fat products (0-0.5% fat) with low protein levels (1-2.5%) normallyneeds addition of a stabilizer to avoid protein sedimentation.

As used herein, the term “bacteriophage” has its conventional meaning asunderstood in the art ie. a virus that selectively infects one or morebacteria. Many bacteriophages are specific to a particular genus orspecies or strain of bacteria. The term “bacteriophage” is synonymouswith the term “phage”. Bacteriophages may include, but are not limitedto, bacteriophages that belong to any of the following virus families:Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae,Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae. Thebacteriophage may be a lytic bacteriophage or a lysogenic bacteriophage.A lytic bacteriophage is one that follows the lytic pathway throughcompletion of the lytic cycle, rather than entering the lysogenicpathway. A lytic bacteriophage undergoes viral replication leading tolysis of the cell membrane, destruction of the cell, and release ofprogeny bacteriophage particles capable of infecting other cells. Alysogenic bacteriophage is one capable of entering the lysogenicpathway, in which the bacteriophage becomes a dormant, passive part ofthe cell's genome through prior to completion of its lytic cycle.

In one embodiment, the lactic acid bacterium according to the presentinvention is resistant to one or more bacteriophage or one or more setsof bacteriophages, in another embodiment, the lactic acid bacteriumaccording to the present invention is resistant to the samebacteriophage that a strain deposited according to the present inventionis resistant to. In the present context, the term “phage robust” is usedinterchangeable with the term “phage resistant”.

In the present context, the term “mutant” should be understood as astrain derived, or a strain which can be derived, from a strain of theinvention (or the mother strain) by means of e.g. genetic engineering,radiation and/or chemical treatment. It is preferred that the mutant isa functionally equivalent mutant, e.g. a mutant that has substantiallythe same, or improved, properties (e.g. regarding texture, shear stress,viscosity, gel stiffness, mouth coating, flavor, post acidification,acidification speed, and/or phage robustness) as the mother strain. Sucha mutant is a part of the present invention. Especially, the term“mutant” refers to a strain obtained by subjecting a strain of theinvention to any conventionally used mutagenization treatment includingtreatment with a chemical mutagen such as ethane methane sulphonate(EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV light, or to aspontaneously occurring mutant. A mutant may have been subjected toseveral mutagenization treatments (a single treatment should beunderstood one mutagenization step followed by a screening/selectionstep), but it is presently preferred that no more than 20, or no morethan 10, or no more than 5, treatments (or screening/selection steps)are carried out. In a presently preferred mutant, less that 5%, or lessthan 1% or even less than 0.1% of the nucleotides in the bacterialgenome have been shifted with another nucleotide, or deleted, comparedto the mother strain. In the present context, the term “variant” shouldbe understood as a strain which is functionally equivalent to a strainof the invention, e.g. having substantially the same, or improved,properties e.g. regarding texture, shear stress, viscosity, gelstiffness, mouth coating, flavor, post acidification, acidificationspeed, and/or phage robustness). Such variants, which may be identifiedusing appropriate screening techniques, are a part of the presentinvention.

In the present context, “texture” is measured as shear stress after 12hours growth at 37 degrees C. The SI unit for shear stress and gelstiffness is pascal (Pa).

An assay to be used for analysis of texture:

The day after incubation, the fermented milk was brought to 13° C. andstirred gently by means of a stick fitted with a bored disc untilhomogeneity of the sample. The rheological properties of the sample wereassessed on a rheometer (StressTech, Reologica Instruments, Sweden)equipped with a C25 coaxial measuring system. The viscometry test wasmade with shear rates varying from 0.27 to 300 1/s in 21 steps. Shearrates were increased and then decreased and the upward and downwardcurves of shear stress and apparent viscosity were recorded. Delay andintegration times were 5 s and 10 s, respectively.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

LEGENDS TO THE DRAWING

FIG. 1 depicts the shear stress of the galactose positive strainCHCC11342 and the galactose positive phage resistant mutant CHCC11977measured with the StressTech rheometer. Shear stress was measured incoagulated milk after over/night growth in milk at 37° C.

FIG. 2 depicts the shear stress of phage resistant mutants of CHCC10019measured with StressTech rheometer. The shear stress was measured incoagulated milk after over/night growth in milk at 37° C.

FIG. 3 depicts the gel stiffness of CHCC12339, phage resistant mutant ofCHCC9204, measured with StressTech rheometer. The gel stiffness wasmeasured in coagulated milk after over/night growth in milk at 37° C.

FIG. 4 depicts the shear stress of CHCC13140, phage resistant mutant ofCHCC5086, measured with StressTech rheometer. The shear stress wasmeasured in coagulated milk after over/night growth in milk at 37° C.

EXPERIMENTAL Example 1 Development of Phage Resistant Streptococcusthermophilus Strains with Improved Texture Properties

Development of CHCC11977

The mother strain CHCC11342 was obtained as described in example 5. Thestrain is a mutant of CHCC6008 and considered to be a galactosefermenting S. thermophilus strain.

The strain CHCC11977 was isolated on M17 agar plate after plating 0.1 mlof an M17 over night culture of CHCC11342 together with 0.1 ml of phageCHPC1152 containing 10E09 (10exp9) phage particles per ml and incubationfor two days at 37° C. One mutant, called CHCC11977, was three timescolony purified and retested in plaque test on M17 agar plates at 37° C.using phage CHPC1152 where phage resistance was confirmed (no singleplaques observed).

The mutant strain was then also tested in M17 broth at 37° C. in thepresence of phage CHPC1152. CHCC11977 remained its phage resistance alsoin liquid culture, whereas CHCC11342, as expected, was attacked byCHPC1152.

CHCC11977 was also tested in milk at different temperatures (without theaddition of infecting phage) showing an acidification activitycomparable to the mother strain CHCC11342.

Analysis of CHCC11342 Texture in Fermented Milk

The day after incubation, the fermented milk was brought to 13° C. andstirred gently by means of a stick fitted with a bored disc untilhomogeneity of the sample. The rheological properties of the sample wereassessed on a rheometer (StressTech, Reologica Instruments, Sweden)equipped with a C25 coaxial measuring system.

The viscometry test was made with shear rates varying from 0.27 to 3001/s in 21 steps. Shear rates were increased and then decreased and theupward and downward curves of shear stress and apparent viscosity wererecorded.

Delay and integration times were 5 s and 10 s, respectively. For furtheranalysis, shear stress at 300 s-1 was chosen.

The rheometer results showed that CHCC11342 had a shear stress value of73.0 Pa compared to CHCC6008 with a shear stress value of 68.0 Pa, seeFIG. 1.

Analysis of CHCC11977 Texture in Fermented Milk

Fermented milk was obtained, and the texture related properties wereanalyzed, as described above for the gal-positive phage resistantmutant.

The rheometer results showed that CHCC11977 had a shear stress valuewhich was furthermore improved by 10% compared to CHCC11342 (shearstress value 80.0 Pa for CHCC11977 compared with 73.0 Pa for motherstrain CHCC11342, see FIG. 1).

Furthermore, the gel stiffness (G*) was increased by 20% for the phageresistant mutant CHCC11977 which showed a value of 126.0 Pa compared toCHCC11342 (gel stiffness 104.0 Pa). With this it was possible to improvethe important rheology parameters shear stress and gel stiffnesssignificantly by isolating a phage resistant mutant from a galactosepositive mother strain.

Sequencing of the galK Promoter Region from CHCC11342

To reveal the type of mutation for the galactose positive mutantCHCC11342 the beginning of the galK gene (coding for the galactokinasefrom S. thermophilus) was sequenced.

For CHCC11342 a mutation in the region of the galK promoter wasidentified (see sequences below). The mutation occurred threenucleotides downstream of the −10-promoter box of the galK gene leadingto a C to A nucleotide shift.

CHCC11342 AAAATA TTGATT TTCCATGTGAAAGGGGT TACGATTTAAGTATAAACAAAAAGAATAAGTGAGATACATC SEQ ID No: 1 CHCC6008 AAAATA TTGATTTTCCATGTGAAAGGGGT TACGAT TTCAGTATAAACAAAAAGAATAAGTGAGATACATCSEQ ID No: 2 AY704368 AAAATA TTGATT TTCCATGTGAAAGGGGT TACGATTTCAGTATAAACAAAAAGAATAAGTGAGATACATC SEQ ID No: 3 Consensus AAAATA TTGATTTTCCATGTGAAAGGGGT TACGAT TTCAGTATAAACAAAAAGAATAAGTGAGATACATCSEQ ID No: 4        −35                    −10                           RBS

Promoter region of the galK gene from CHCC11342. The point mutationwithin the CHCC11342 galk promoter region is indicated with grey colorcode. The published galK sequence from S. thermophilus ST111 (Genbankaccession no. AY704368) is indicated for comparison. −35: −35-promoterbox; −10: −10-promoter box; RBS: ribosome binding site.

Further Phage Resistant Mutants

To reveal a possible relationship between phage resistance and galactosepositive phenotype five additional phage resistant mutants were isolatedon M17 galactose agar plates from strain CHCC6008 (gal−) which is themother strain of CHCC11342 (gal+). All five phage resistant mutants,CHCC11396, CHCC11397, CHCC11398, CHCC11399, and CHCC11340 (resistant tophage CHPC1152) were unable to ferment galactose meaning the gal+phenotype is not directly related with phage resistance. On the otherhand it was demonstrated by plaque assay that CHCC11342 (gal+) was stillsensitive to phage CHPC1152.

Example 2 Development of Phage Resistant Lactobacillus delbrueckiisubsp. bulgaricus Strains with Improved Texture Properties.

From the mother strain CHCC10019 (DSM19252), phage resistant mutantswere isolated as follows:

Mutants were picked from MRS agar plates containing 10 mM CaCl₂/10 mMMgCl₂ after plating 0.1 ml of an MRS over night culture of CHCC10019together with 0.1 ml of a CHPC658 phage lysate containing 10E06 phageparticles per ml and anaerobic incubation for two days at 37° C. Thirtymutants were isolated and tested in cross-streaking towards phageCHPC658. Twenty-nine mutants appeared resistant in the cross-streakingtest, and were afterwards three times colony-purified on MRS agar platesat 37° C.

The 29 mutants were tested in microtiter plates for acidificationprofile and phage resistance. Two microtiter plates were prepared withmilk and each plate was inoculated with 2% of the respective mutant. Forone plate, 2% peptone-salt diluent (control) was added to each well, andto the other microtiter plate 2% CHPC658 containing 10E06 phageparticles per ml was added. The two plates were incubated at 37° C. fortwo days, and pH of each well was recorded every 12 minutes. All mutantswere phage resistant compared to the mother strain CHCC10019, which wasattacked by phage CHPC658.

Twelve mutants were chosen based on the acidification profile in MRS andmilk (similar to the mother strain) and the viscosity of the strains.

Analysis of CHCC10019 Phage Resistant Mutants for Texture Properties inFermented Milk

After incubating the twelve mutants and mother strain CHCC10019 overnight in milk at 37° C., the fermented milk was adjusted to 13° C. andstirred gently by means of a stick fitted with a disc containing holesuntil homogeneity of each sample. The rheological properties of eachsample was assessed on a rheometer (StressTech, Reologica Instruments,Sweden) equipped with a C25 coaxial measuring system.

The viscometry test was made with shear rates varying from 0.27 to 3001/s in 21 steps. Shear rates were increased and then decreased and theupward and downward curves of shear stress and apparent viscosity wererecorded. Delay and integration times were 5 s and 10 s, respectively.For further analysis, shear stress at 300 s-1 was chosen.

The results from the rheology measurements showed that all twelvemutants (such as CHCC12813 and CHCC12841) had an improved viscosity,measured as shear stress, compared to CHCC10019. The highest shearstress value was obtained for CHCC12841 (74.0 Pa) whereas CHCC10019 hada shear stress value of 58.0 Pa, see FIG. 2. On the figure, the rheologydata for the twelve phage resistant mutants is shown. All mutants havean increase in shear stress ranging from 10% up to 28% compared toCHCC10019.

Based on acidification activity and rheology data when the mutants weregrown in co-culture with other strains CHCC12813 was selected as mostpromising candidate of the isolated mutants for further applicationtests.

This experiment demonstrates that the important rheological parametershear stress of a bacterial strain will be significantly improved byisolating phage resistant mutants of the strain.

Example 3 Isolation of Phage Resistant Mutants of Streptococcusthermophilus Strain CHCC9204

From the mother strain CHCC9204, registered in Chr. Hansen culturecollection, a phage resistant mutant was isolated. The mutant wasisolated on M17-2% lactose agar plates with 10 mM MgCl₂/CaCl₂ afterplating 0.1 ml of an M17 lactose over night culture of CHCC9204 togetherwith 0.1 ml of phage CHPC1057 containing 1×10exp09 phage particles perml and incubation overnight at 37° C.

Among several mutants one strain, called CHCC12339, was three timescolony purified and retested in plaque test on M17 lactose agar platesat 37° C. using phage CHPC1057 for phage challenge, and phage resistancewas confirmed (no single plaques observed in plaque test). CHCC12339 wasalso tested in milk showing an acidification activity comparable to themother strain.

Analysis of Texture Properties of Phage Resistant Mutant CHCC12339 inRermented Milk

After mutant CHCC12339 and mother strain CHCC9204 were incubated overnight in milk at 37° C., the fermented milk was brought to 13° C. andstirred gently by means of a stick fitted with a bored disc untilhomogeneity of each sample. The rheological properties of each samplewas assessed on a rheometer (StressTech, Reologica Instruments, Sweden)equipped with a C25 coaxial measuring system.

The viscometry test was made with shear rates varying from 0.27 to 3001/s in 21 steps. Shear rates were increased and then decreased and theupward and downward curves of shear stress and apparent viscosity wererecorded. Delay and integration times were 5 s and 10 s, respectively.For further analysis, shear stress at 300 s-1 was chosen.

The results from the rheology measurements showed that mutant CHCC12339leads to an increase of gel stiffness (G*) by 22% compared to CHCC9204,see FIG. 3. On the figure, the gel stiffness values (G*) for the phageresistant mutant CHCC12339 (78.0 Pa) and mother strain CHCC9204 (64.0Pa) are compared.

Example 4 Isolation of Phage Resistant Mutants of Streptococcusthermophilus Strain CHCC5086

From the mother strain CHCC5086, registered in Chr. Hansen culturecollection, a phage resistant mutant was isolated.

The mutant was isolated on M17-2% lactose agar plates with 10 mMMgCl₂/CaCl₂ after plating 0.1 ml of an M17-2% lactose over night cultureof CHCC5086 together with 0.1 ml of phage CHPC1089 containing 1×10exp08phage particles per ml and incubation overnight at 37° C.

Among several mutants one strain, called CHCC13140, was three timescolony purified and retested in plaque on M17 lactose agar plates at 37°C. test using phage CHPC1089 for phage challenge, and phage resistancewas confirmed (no single plaques observed in plaque test). CHCC13140 wasalso tested in milk acidification test showing an acidification activitycomparable to the mother strain.

Analysis of Texture Properties of Phage Resistant Mutant CHCC13140 inFermented Milk

After mutant CHCC13140 and mother strain CHCC5086 were incubated overnight in milk at 37° C., the fermented milk was brought to 13° C. andstirred gently by means of a stick fitted with a bored disc untilhomogeneity of each sample. The rheological properties of each samplewas assessed on a rheometer (StressTech, Reologica Instruments, Sweden)equipped with a C25 coaxial measuring system.

The viscometry test was made with shear rates varying from 0.27 to 3001/s in 21 steps. Shear rates were increased and then decreased and theupward and downward curves of shear stress and apparent viscosity wererecorded. Delay and integration times were 5 s and 10 s, respectively.For further analysis, shear stress at 300 s-1 was chosen.

The results from the rheology measurements showed that mutant CHCC13140leads to an increase of shear stress by 14% compared to CHCC5086, seeFIG. 4. On the figure, the shear stress data for the phage resistantmutant CHCC13140 (shear stress 33.0 Pa) and mother strain CHCC5086 (28.0Pa) are compared.

Example 5 Preparation of a Galactose Positive Mutant of a StreptococcusStrain

General Method for Obtaining Gal+ Strains

Prior to the mutant isolation the mother strain (e.g. CHCC6008) werestreaked on M17 agar plates with 2% galactose (M17-gal plates). CHCC6008did not grow on galactose as sole carbohydrate source, and thus themother stain is considered to be gal−.

Over night cultures of the mother strain were then plated on M17-galplates and several colonies could be isolated after two days of growthat 37° C.

Several mutants were purified on M17-gal plates and retested in M17broth containing 2% galactose as sole carbohydrate.

A mutant may be obtained by means of e.g. genetic engineering, radiationand/or chemical treatment, or the mutant may be a spontaneous mutant.

A mutant was considered galactose positive when the pH was reduced by avalue of at least 1.0 after 16 hours incubation at 37 degrees C. in M17with 2% galactose (galactose added as sole carbohydrate), inoculated inan amount of at least 10E4 cells pr ml of medium.

Whereas CHCC6008 did not lower the pH in M17-gal broth significantly,CHCC11342, one of the purified mutants, reached a pH of 5.4 after 16hours at 37° C., and was therefore considered a galactose-fermenting(gal+) mutant of CHCC6008.

Isolation of Galactose Fermenting Strains

Mutants were isolated as galactose fermenting mutant of S. thermophilusstrain CHCC6008 (=ST6008, DSM18111). The CHCC6008 cells were neithermutagenized with any mutagenic compound nor by UV light prior to themutant isolation step. The isolated strains resemble thereforespontaneous galactose positive mutants of CHCC6008.

Prior to the mutant isolation CHCC6008 was streaked on M17 agar plateswith 2% galactose (M17-gal plates). CHCC6008 did not grow on galactoseas sole carbohydrate source.

Over night culture of CHCC6008 were then plated on M17-gal plates andseveral colonies could be isolated after two days of growth at 37° C.Several mutants were purified on M17-gal plates and retested in M17broth containing 2% galactose as sole carbohydrate.

Whereas CHCC6008 did not lower the pH in M17-gal broth significantly,CHCC11379, one of the purified mutants, reached a pH of 5.3 after 10hours at 37° C., and was therefore considered a galactose-fermentingmutant from CHCC6008.

Strains CHCC11342 and CHCC11976 were isolated the same way.

Isolation of Mutants by Genetic Engineering

Galactose positive mutants can also be generated by site directedmutagenesis. Oligonucleotides carrying the mutated nucleotide within thegalK −10 promoter box is used to amplify a specific DNA fragment by PCR.The PCR fragment carrying the desired mutation is cloned into a vectorplasmid and transformed into the S. thermophilus target strain, and themutation is integrated into the chromosome and exchanging the wild typegalK promoter region by recombination. Isolation of strains is done asabove.

Analysis of Texture in Fermented Milk

The day after incubation, the fermented milk was brought to 13° C. andstirred gently by means of a stick fitted with a bored disc untilhomogeneity of the sample. The rheological properties of the sample wereassessed on a rheometer (StressTech, Reologica Instruments, Sweden)equipped with a C25 coaxial measuring system.

The viscometry test was made with shear rates varying from 0.27 to 3001/s in 21 steps.

Shear rates were increased and then decreased and the upward anddownward curves of shear stress and apparent viscosity were recorded.

Delay and integration times were 5 s and 10 s, respectively. For furtheranalysis, shear stress at 300 s-1 was chosen. The rheometer resultsshowed that CHCC11379 had a shear stress which was improved by 10%compared to CHCC6008 (shear stress value 74.0 Pa (Pascals) for CHCC11379compared with 67.5 Pa for mother strain CHCC6008.

Analysis of Texture in Milk

In another experiment CHCC11379 was used as a part of a yoghurt culturewhere strains from S. thermophilus are co-cultured in skimmed milk at43° C. together with a strain from the species Lactobacillus delbrueckiissp. bulgaricus. When the only difference in the production of yoghurtwas the use of CHCC11379 instead of wild type CHCC6008 the shear stresswas also increased by 10% (105.0 Pa for the yoghurt culture containingCHCC11379 compared with 94.7 Pa for the yoghurt culture containingCHCC6008).

Sequencing of the galK Promoter Region from CHCC11379

To reveal the type of mutation for the gal positive mutant CHCC11379 thebeginning of the galK gene (coding for the galactokinase from S.thermophilus) was sequenced.

For CHCC11379 a mutation in the region of the galK promoter wasidentified (see below). The respective mutation will most likely lead toa stronger promoter activity compared to the mother strain 6008,explaining the observed gal-positive phenotype. This is based on thefact that the consensus sequence for the −10-promoter box is “TATAAT”,and that a mutation at nucleotide 3 of the −10 box (region) for CHCC6008(“TACGAT”) leads to a −10 box with a higher similarity to the consensussequence in CHCC11379 (“TATGAT”).

CHCC11379 AAAATA TTGATT TTCCATGTGAAAGGGGT TATGATTTCAGTATAAACAAAAAGAATAAGTGAGATACATC SEQ ID NO: 5 CHCC6008 AAAATA TTGATTTTCCATGTGAAAGGGGT TACGAT TTCAGTATAAACAAAAAGAATAAGTGAGATACATCSEQ ID NO: 2 AY704368 AAAATA TTGATT TTCCATGTGAAAGGGGT TACGATTTCAGTATAAACAAAAAGAATAAGTGAGATACATC SEQ ID NO: 3 Consensus AAAATA TTGATTTTCCATGTGAAAGGGGT TACGAT TTCAGTATAAACAAAAAGAATAAGTGAGATACATCSEQ ID NO: 4       −35                    −10                           RBS

Promoter region of the galK gene from CHCC11379. The point mutationwithin the CHCC11379 galk promoter is indicated with grey color code.The published galK sequence from S. thermophilus ST111 (Genbankaccession no. AY704368) is indicated for comparison. −35: −35-40promoter region; −10: −10-promoter region; RBS: ribosome binding site.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Deposits and Expert Solution

The strains Streptococcus thermophilus CHCC11977 and CHCC11342 weredeposited at DSMZ (Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) underthe accession numbers DSM22935 and DSM22932, resp., on Sep. 8, 2009.CHCC6008 has been deposited at DSMZ under the accession number DSM 18111on Mar. 29, 2006.

Bacteriophages CHPC658, CHPC1057 and CHPC1152 were deposited at DSMZ on27 Aug. 2010, and given the deposit numbers DSM23961, DSM23962, andDSM23994, resp.

Further deposits at DSMZ:

CHCC10019 (DSM19252) and CHCC3984 (DSM19251): Date of deposit 3 Apr.2007,

CHCC2008 (DSM22584) and CHCC5086 (DSM22587): date of deposit 19 may2009,

CHCC11379 (DSM22884): date of deposit 26 Aug. 2009,

CHCC11976 (DSM22934): date of deposit 8 Sep. 2009;

CHCC13140 (DSM 24023), CHCC12813 (DSM24074), and CHPC1089 (DSM 24022):date of deposit 29 Sep. 2010;

CHCC12339 (DSM24090): date of deposit 14 Oct. 2010.

Streptococcus thermophilus CHCC5086 (DSM22587): date of deposit: 19 May2009.

CHCC9204 (DSM19243): date of deposit 29 Mar. 2007.

The deposits have been made under the conditions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure.

The Applicant requests that a sample of the deposited microorganismsshould be made available only to an expert approved by the Applicant.

References

Appl. Environ. Microbiol. 71,7, p. 3659-67 (2005);

International Journal of Food Microbiology 113 (2007) 195-200;

Applied And Environmental Microbiology, Feb. 2002, p. 784-790;

J Dairy Sci 92: 477-482 (2009)

WO2008/040734A1, WO2007/025097A2, US7241610B2, WO2007/144770A2,WO2004/085607A, WO2008/148561A, WO11000879A, WO11000883A, WO10023178A

All references cited in this patent document are hereby incorporatedherein in their entirety by reference.

The invention claimed is:
 1. A bacterial strain belonging to the speciesStreptococcus thermophilus, selected from the group consisting ofdeposited strains CHCC11977 (DSM22935), CHCC12339 (DSM24090), CHCC13140(DSM 24023), and mutants or variants derived from any one of saiddeposited strains and having all the identifying characteristics of thedeposited strain.
 2. A bacterial strain belonging to the speciesLactobacillus delbrueckii subsp. bulgaricus, selected from the groupconsisting of deposited strain CHCC12813DSM24074), and mutants orvariants derived therefrom and having all the identifyingcharacteristics of the deposited strain.
 3. A composition comprising thebacterial strain of claim
 1. 4. A method for producing a fermented milkproduct, comprising fermenting a milk substrate with a lactic acidbacterium or strain of claim
 1. 5. A composition comprising thebacterial strain of claim
 2. 6. The bacterial strain of claim 1, whereinthe bacterial strain is a mutant or variant derived from one of saiddeposited strains that is more resistant to phage than the mother strainof said deposited strain.
 7. The bacterial strain of claim 1, whereinthe bacterial strain is a mutant or variant derived from one of saiddeposited strains that generates an increased shear stress in fermentedmilk compared to the mother strain of said deposited strain, measuredafter 12 hours of growth at 37° C. when inoculated in an amount of atleast 10E4 cells/ml milk.
 8. The bacterial stain of claim 7, wherein theshear stress in fermented milk is increased by at least about 10%compared to the mother strain of said deposited strain.
 9. The bacterialstrain of claim 2, wherein the bacterial strain is a mutant or variantderived from one of said deposited strains that generates a shear stressin fermented milk in the range from 60 to 100 Pa, measured after 12hours of growth at 37° C. when inoculated in an amount of at least 10E4cells/ml milk.
 10. A method for producing a fermented milk product,comprising fermenting a milk substrate with a lactic acid bacterium orstrain of claim 2.